Project Summary/Abstract The long-term objective of this application is to fully understand the biology of endothelial laminin in cerebrovascular development and function in physiological conditions. This is in line with NHLBI?s overarching objective to understand normal biological function. This proposal aims to investigate the biological function of endothelial laminin in blood brain barrier (BBB) integrity under homeostatic conditions, and explore its underlying molecular mechanism. In Aim 1, whether loss of endothelial laminin affects BBB formation in early development or BBB maintenance at later stage will be investigated using an innovative endothelial laminin knockout mouse line (EKO) generated in our laboratory. The extent of BBB disruption will be determined using fluorescent tracers of various molecular weights. Next, the underlying molecular mechanism (paracellular and/or transcellular transport) will be explored at biochemical, ultrastructural, and functional levels both in vitro and in vivo. Since hematopoietic cell-derived laminin is also ablated in these mutants, we will further investigate if BBB breakdown in EKO mice is due to loss of laminin in endothelial cells or hematopoietic cells using the VE-Cadherin-CreERT line. In Aim 2, the hypothesis that endothelial laminin regulates BBB integrity via adenylyl cyclase-2 (AC2) will be tested. First, whether and how exactly endothelial laminin regulates AC2 expression in brain microvascular endothelial cells will be examined in vitro and in vivo. Next, the function of AC2 in BBB permeability and paracellular/transcellular transport will be investigated using both loss-of-function and gain-of-function approaches. Third, whether the effect of AC2 on BBB integrity relies on its adenylyl cyclase activity (generation of cAMP) will be investigated using AC2 inhibitors and/or activators. If the answer is yes, which cAMP-downstream effector/signaling pathway (PKA versus Epac) mediates AC2?s BBB-regulating function will be explored using PKA- and/or Epac-specific inhibitors and activators. Successful completion of this proposal will elucidate the biological function of endothelial laminin in BBB integrity and its underlying molecular mechanism; uncover a previously unrecognized role of AC2 in transcellular transport (transcytosis); and identify endothelial laminin and AC2 as novel molecular targets in BBB regulation, which will promote the development of innovative treatments for BBB disruption. In addition, this proposal will also generate innovative research materials (e.g. transgenic mouse line and lentiviral constructs) useful in the field of laminin/basement membrane, which is understudied due to its intrinsic complexity and lack of research tools.